Hello there, I am trying to isolate protoplast from Leptosphaeria biglobosa for a transfection experiment I am planning to do but so far after incubating my mycelia for 6 hours in 10ml of KCL/citric acid containing cellulase and trichoderma harzianum lysing enzyme, I end up with a very small amount of protoplast.
So I was wondering to increase the volume of the enzymes or decrease the volume of the mycelia used. Any help please I am currently using the protocol adapted from Szewczyk et al., 2007.
Thank you
Dear Mr. Filippou: Your protocol is correct but the enzyme for generating the protoplast from your fungus Leptosphaeria biglobosa may not be correct. We normaly isolate the cell wall from 30 - 40 hr grown Leptosphaeria biglobosa in a liquid medium as per the standard protocol. Use this fungal cell wall as carbon source in the medium and inoculate with T. harzianum (36 hr grown in liquid medium) as inoculum. Allow the fungusT/ harzianum to grow for 72 - 96 hr on a rotary shaker at approx. 100 rpm at appropriate temperature. After the incubation, centrifuge the broth and use the supernatant as enzyme to get protoplast from freshly grown (32 - 36 hr) Leptosphaeria biglobosa. You may use lysing enzyme available with you along with freshly prepared enzyme.
If problem connected with your enzyme activity you can find out it by increasing of enzyme amount or decreasing of substrate concentration.
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